Estrogens are essential regulators of many physiological processes including maintenance of the female sexual organs, the reproductive cycle and numerous neuroendocrine functions. Importantly, these hormones also play crucial roles in several disease states, particularly in hormone-dependent cancers, such as breast, endometrial, pancreatic, and ovarian carcinomas. Worldwide, breast cancer is the leading cause of death from cancer among women, with more than 500,000 estimated to have died from the disease in 2009. Furthermore, systemic estrogen levels are key indicators of breast cancer recurrence [Breast Cancer, 15:270-277 (2008)], implicating estrogen in the progression of debilitating and life-threatening diseases.
Through binding to their target estrogen Receptor (ER), estrogen promotes both cell proliferation as well as tumor invasiveness [Genome Biol., 5(9): R66 (2004)]. Given that the majority of breast, endometrial, and ovarian carcinomas express ERs and are thus potentially sensitive to the growth promoting effects of estrogens, current treatment strategies for ER+ cancers are largely concerned with the inhibition of the estrogen synthesis pathway.
Recent studies have demonstrated that estrogens are produced in ER+ cancers by several enzymes. Among these enzymes in the estrogen biosynthetic pathway, aromatase is considered a crucial enzyme for estrogen synthesis. High concentrations of circulating inactive steroids, such as androstenedione, are major precursors to intratumoral estrogen production.
Aromatase is a member of the P450 mono-oxygenase family and is directly responsible for the conversion of androgens (androstenedione and testosterone) to estrogens (estrone and estradiol, respectively), through oxidative removal of the C19 methyl group and subsequent aromatization. A majority of ER+ cancers have aromatase activities that are comparable with or greater than those found in other tissues, and aromatase mRNA levels are typically increased in ER+ carcinomas relative to nonmalignant tissue [J. Clin. Endocrinol. Metab., 81:2344-9 (1996); Endocr. Relat. Cancer., 12:701-20 (2005)]. For example, it has been shown that the gene for aromatase is over expressed in >75% of breast cancer lines, which results in the disease tissue being saturated in estrogens, promoting the growth of the cancerous cells. Not only are expression levels increased in ER+ cancer cells, but they are also increased in surrounding cells, such as adipose tissue and stromal cells [Hum. Pathol., 5:530-3 (1994); J. Clin. Endocrinol. Metab., 79: 627-32 (1994); J. Steroid Biochem. Mol. Biol., 95: 35-9 (2005)]. Furthermore, aromatase mRNA levels have been shown to correlate with the level of breast tumor invasiveness; where they are highest in invasive carcinoma, modest in noninvasive carcinoma, and lowest in normal tissue [Endocr. Relat. Cancer., 15: 113-24 (2008)]. Thus, the aromatase enzyme is a key target in the inhibition of the estrogen biosynthesis pathway. Inhibition of aromatase has been demonstrated to significantly reduce re-growth of certain cancer after initial treatments [Lancet Oncol., 9(1):8-10 (2008)].
Aromatase inhibitors are an established alternative treatment option to other forms of estrogen regulation [J. Steroid Biochem. Mol. Bio., 95: 75-81 (2005)], including molecules such as tamoxifen that are known to inhibit estrogen signaling [Mol Endocrinol. 9: 659-669 (1995); Nature 387: 733-736 (1997)]. Substances that have been reported to at least partially inhibit aromatase action include, for example, Δ1-testololactone (U.S. Pat. No. 2,744,120), 4-hydroxyandrost-4-ene-3,17-dione and esters thereof (for example, U.S. Pat. No. 4,235,893), 10-(1,2-propadienyl)-estr-4-ene-3,17-dione (U.S. Pat. No. 4,289,762), 10-(2-propynyl)-estr-4-ene-3,17-dione [J. Am. Chem. Soc., 103: 3221 (1981) and U.S. Pat. No. 4,322,416], 19-thioandrostene derivatives (Europ. Pat. Appl. 100566), androsta-4,6-diene-3,17-dione, androsta-1,4,6-triene-3,17-dione (G.B. Pat. Appl. 2,100,601A), androsta-1,4-diene-3,17-dione [Cancer Res., 42: 3327 (1982)], 6-substituted androsta-1,4-diene-3,17-diones (U.S. Pat. No. 4,808,616), substituted androsta-1,4-diene-3,17-diones (U.S. Pat. No. 4,904,650), alpha-heterocyclic substituted tolunitriles (U.S. Pat. Nos. 4,978,672; 5,112,845; 5,352,795; 5,473,078), tetrazolyl substituted benzonitriles (U.S. Pat. No. 5,073,574), diaryl methanes (U.S. Pat. No. 5,426,196), 4-[alpha(cyanophenyl)-1-(1,2,3-triazolyl)methyl]-benzonitrile (U.S. Pat. No. 5,457,209), heterocyclic diarylalkyl compounds (U.S. Pat. Nos. 5,703,109; 5,962,495). Although several of these compounds are able to inhibit aromatase, their binding activities are not specific; as a result, they cause a number of off target effects in patients [Expert Opin. Pharmacother., 10(9):1435-47 (2009)].
Certain compounds have been found to interact with heightened specificity to the aromatase enzyme. Exemestane (6-methylenandrost-1,4-diene-3,17-dione), which is described in U.S. Pat. No. 4,808,616, is structurally related to the natural substrate androstenedione and is initially recognized by the aromatase enzyme as a false substrate thus competing with androstenedione at the active site of the enzyme. The compound is transformed to an intermediate that binds irreversibly to the enzyme, causing its inactivation (also known as suicide inhibition). Although exemestane is more selective than its predecessors in the inhibition of aromatase, its use has also been shown to result in potentially debilitating off target effects. For example, some studies have demonstrated that the sex hormone binding globulin, a protein that regulates the bioavailability of sex hormones, is dysregulated in patients taking exemestane [Clin. Breast Cancer, 9(4): 219-24 (2009)]. Yet other studies have demonstrated that Exemestane can interfere with calcium homeostasis [Curr. Oncol. Rep., 6(4): 277-84. (2004); The Lancet Oncology, 8(2): 119-127 (2007); Clin. Breast Cancer, 8(6): 527-32 (2008)]. Most importantly, however, is that exemestrane use has been associated with additional side effects, such as severe hepatotoxicity, that are not observed with other aromatase inhibitors [Breast Cancer Res. Treat., October 16. (Epub ahead of print) (2009)].
Additional compounds related to exemestane, include (S)-6-methyloxaalkyl exemestane derivatives (PCT Appl. No. WO 2007041564), fluorinated 4-aminoandrostadienones (PCT Appl. No. WO 9419365), fluorinated 6-methyleneandrosta-1,4-dien-3-ones (PCT Appl. No. WO 9501366), and androst-4-eno[4,5-b]pyrroles (PCT Appl. No. WO 9404554). A range of 6-alkoxy and 6-keto steroids have been synthesized by reaction of an unsubstituted steroid with a suitable lower alcohol such as methanol or ethanol together with a cupric halide, where the halide can be either chloride or bromide (U.S. Pat. No. 3,032,565). Steroid derivatives were also synthesized by reaction of the epoxy derivatives with a compound such as sodium thiocyanate and subjecting the reaction product to a dehydration reaction (Japanese Pat. No, 63-045294, 1998). A range of 6 and 7 substituted androst-1,4-diene derivatives were obtained by converting a suitable bromo derivative to the corresponding thiol followed by further alkylation or acylation (Japanese Pat. No., 07-215992). A range of C6 substituted androst-4-ene-3,17-diones were formed by bringing the unsubstituted precursor andros-4-ene-3,17-dione into contact with certain microorganisms (Japanese Pat. No. WO 1988/05781).
In probing the binding pocket of the active site of aromatase, Numazawa et al. synthesized and tested a range of 6-ester and 6-ether substituted androst-4-ene-3,17-diones as well as their 1,4-diene and 1,4,6-triene analogues. The 6β-methoxy and 6β ethoxy androsta-1,4-diene-3,17-dione derivatives were found to be suicide substrates of aromatase (M. Numazawa, M Ando and R. Zennyoji, J Steroid Biochem Molec Biol. 2002, 82:65-73 and Numazawa et al., Biochem J., 1998, 3299(1), 151-156).
In their evaluation of 4-substituted-4-androstene-3,17-dione derivatives as, Abul-Hajj et al., showed that aromatase has a hydrophobic pocket in the active site around the C4α region of androstenedione. (Y. J. Abul-Hajj, X-P, Liu and M. Hedge, J Steroid Biochem Molec Biol. 1995, 54:111-119). Marsh et al. showed that esterification of the 4-hydroxy analogues generally reduced activity but conjugation of the 3-keto 4-ene system to produce 4-hydroxy-4,6-androstadiene-3,17-dione caused more rapid inactivation of aromatase in rat ovarian microsomes than the 4-hydroxyandrostenedione (D. A. Marsh, H. J. Brodie, W. Garrett, C-H, Tsai-Morris and A. M. H. Brodie, J. Med. Chem., 1985, 28:788-795). A variety of ester and ether derivatives of 4-hydroxy 4-androstenedione were suggested as means of regulating athletic function in humans. (U.S. Pat. No. 6,586,417 B1).